This invention relates to a transgenic fish carrying a plasmid-based marker, and in particular relates to a transgenic fish for use in evaluating the effect of a potential mutagen. The transgenic fish is exposed to the mutagen, and mutagenesis is detected by assaying for a mutation target nucleic acid sequence present as a genomically integrated transgene in the transgenic fish.
The health risk posed by exposure to mutagenic agents in the environment remains an important concern as it is known that induction of mutations may lead to various somatic or inherited diseases. In particular, cancer has been shown to result from a series of mutations in specific oncogenes and tumor suppressor genes (Vogelstein et al., N. Engl. J. Med. 319: 525-532 (1988)). Despite the recognition of the role of induced mutation as an important event leading to disease, there are few methods available for the assessment of genetic hazard, or focus on the study of gene mutations as they occur at the DNA level in vivo. As a result, there is an immediate need to develop sensitive and biologically relevant methods that can be applied to the study of the mechanisms of mutagenesis and hazard assessment.
There are two practical requirements common to any study of mutagenesis: 1) the specific loci to be examined should be sensitive to mutation induction, and 2) the mutants must be recovered in sufficient numbers. Until recently, progress in the analysis of gene mutations directly at the DNA level was limited by the standard molecular techniques and the available endogenous genes. During past volts, the most relevant assays for induction of transmissible mutations have been based on the appearance of visible or biochemical mutations among the offspring of exposed mice (L. B. Russell et al., Mutation Res. 86: 329-354 (1981); L. R. Valcovic et al., Environ. Health Perspect. 6:201-205 (1973); S. E. Lewis et al., Prog. Clin. Biol. Res. 209B. 359-365 1986)). These tests cannot be practically applied to large numbers of compounds because they require extensive resources and very large numbers of animals. The tests also fail to provide information regarding somatic mutagenesis or clustering of mutations, which may be important in the understanding of the development of various diseases.
In order to circumvent some of the problems inherent in rodent assays, short-term mutagenicity tests were developed, based on the assumption that many of the chemicals toxic to rodents would also be genotoxic to bacteria. However, an analysis by the National Toxicology Program (R. W. Tennant et al., Science 236:933-941 (1987)) revealed significant differences in results between rodent and bacterial tests. This failure of predictive correlation may be related to: 1) a lack of understanding of the roles mutation plays in cell transformation, and 2) differences between animals and bacterial cells in terms of exposure, biological milieu, metabolism, replication and repair. While comparisons between animals and animal cells in culture provide appropriate genomic similarity, there are few known biological markers for mutation of cells in culture. The biological markers that have been identified are restricted to specific cell types and therefore are of limited use for in vivo comparisons.
There thus remains a need to combine the simplicity of short-term in vitro assays with in vivo studies. Ultimately, reliable and realistic hazard assessment and informative mechanistic studies of mutagenesis require the development of practical methods for evaluating somatic and genetic events in whole animals exposed to environmental agents. New approaches that use recombinant DNA and gene transfer techniques to develop transgenic animal models offer significant promise for in vivo studies of mutagenesis, cancer, birth defects and other diseases (T. L. Goldsworthy et al., Fund. Appl. Toxicol. 22:8-19 (1994)). Transgenic rodents that carry genes specifically designed for the quantitation of spontaneous and induced mutations are currently available and represent a major advance in the study of mutagenesis by allowing rapid analysis of tissue-specific mutations in a whole organism following mutagenic agent exposure (J. C. Mirsalis et al., Ann. Rev. Pharmacol. Toxicol. 35:145-164 1995)).
To be effective, the transgenic approach as applied to mutagenesis should include the following components: 1) unique genes with known sequences; 2) a capacity to observe changes at the single gene copy level; 3) an easily attainable sample population of sufficient size to allow measurement of low frequency events; and 4) the ability to determine the exact nature of the mutation, independent of the host phenotype. Transgenic mutagenesis assay systems based on this approach rely on bacteriophage or plasmid shuttle vectors to carry a mutation target. The basic principle in this approach is that a recombinant gene which carries a mutation target (shuttle vector) is introduced into a host genome. Following exposure to a mutagen, the target gene is recovered to serve as an indicator of mutagenesis (reviewed by R. B. Dubridge et al., Mutagenesis 3(1):1-9 (1988)).
Shuttle vectors currently in use include both bacteriophage-based and plasmid-based vectors. For example, the lambda (xcex) bacteriophage-based recombinant vector combines cos site packaging for recovery of the phage sequence from the host DNA and uses the lacI, lacZ or cII genes as the target gene (J. S. Lebkowski et al., Proc. Natl. Acad. Sci. 82:8606-8610 (1985); J. A. Gossen et al., Proc. Natl. Acad. Sci. 86:7971-7975 (1989); J. L. Jakubczak et al., Proc. Natl. Acad. Sci. U.S.A., 93:9073-9078 (1996)). Another system is based on the pUR288 plasmid vector which contains the lacZ sequence as the mutation target (M. Boerrigter et al., Nature 377:657-659 (1995); M. Dollxc3xa9 et al., Mutagenesis 11:111-118 (1996)). In both the xcex and plasmid-based assays, mutation-induced inactivation of the lac genes are then detected histologically in E. coli. Another system is based on the bacteriophage xcfx86X174 integrated shuttle vector in which the vector is recovered by transfection. This vector is recovered from the transgenic host, transfected into a suitable E. coli host, and mutations at specific locations in the phage sequence are identified by suppressor-mediated selection on permissive and non-permissive E. coli (H. V. Malling et al., Mutation Res. 212:11-21 (1989); R. N. Winn et al., Marine Environ. Res. 40(3):247-265 (1995)).
A fundamental limitation of the bacteriophage-based mutation detection systems is their apparent inability to detect large-scale DNA deletions characteristically induced by clastogenic agents such as ionizing radiation (K. Tao et al., Proc. Nat""l. Acad. Sci, U.S.A., 90:10681-10685 (1993)). Most deletions reported thus far in the abased systems have only been 1-23 base pairs in length. Deletions in the range of hundreds of base pairs are rarely reported using bacteriophage-based mutagenesis detection assays (G. Douglas et al., Mutagenesis 9:451-458 (1994)). Current estimates, depending upon the particular test system, are that up to 90% of radiation-induced mutations are thought to be DNA deletions. The bacteriophage shuttle-vector systems seem to have an inherent bias against detecting certain types of deletions primarily due to restrictive packaging and recovery requirements. It is speculated that since two intact cos-sites are required for the packaging of a single xcex vector, any deletions that extend into regions adjacent to a transgene concatamer may prevent vector recovery.
A plasmid-based process for detecting mutations in whole animals is described in Gossen et al. (U.S. Pat. No. 5,602,300), but is limited to use in transgenic mammals, namely rodents. The plasmid pUR288, which contains a pBR322 Ori for replication, the ampicillin gene, and the whole lacZ gene including the lacZ operator sequence, was inserted into a bacteriophage lambda vector and transferred to the germ line of a mouse by means of microinjection of fertilized egg cells. The lacz-containing plasmid was purified from chromosomal DNA of a resulting transgenic mouse by treating the genomic DNA with a restriction enzyme (also known as a restriction endonuclease), then contacting the restriction digest to a solid support comprising LacI repressor protein (i.e., a lacZ operator binding material) to bind and isolate the plasmid. Gossen et al. (Mut. Res. 331:89-97 (1995)) also disclose incorporation of the linearized form of the plasmid directly into mammalian DNA (without using a bacteriophage vector); and detection of mutations in the lacZ gene by plasmid rescue as well as bacteriophage rescue. Vijg et al. (U.S. Pat. No. 5,817,290) teach a similar plasmid-based method for detection of mutations, except that the plasmid vector is recovered by simultaneously excising it from genomic DNA and contacting it with the solid affinity suppport.
There remains a need for an improved mutagenesis assay detection system capable of detecting mutagenic events that may be missed by bacteriophage-based systems. Additionally, alternative animal models are needed to extend and improve methods used to assess the potential genetic health risks posed by exposure to mutagens the environment.
A transgenic fish has been developed for use in a plasmid-based mutagenesis detection system. The system allows in vivo quantitation of By spontaneous and induced mutations using a recoverable mutation target nucleic acid sequence and assay system. After exposure of the transgenic fish to a mutagen, DNA is extracted from the fish tissue, and the mutation target nucleic acid sequence is recovered and analyzed for mutagenesis, preferably using a bioassay in indicator bacteria. In a preferred embodiment, the mutagenesis detection system of the invention is based on the pUR288 plasmid.
The transgenic fish of the invention is one whose somatic and germ cells contain at least one genomically integrated copy of a plasmid carrying an assayable mutation target nucleic acid sequence. The plasmid is preferably one that is suitable for cloning into E. coli. The invention further provides a transgenic fish gamete, including an transgenic fish egg or sperm cell, a transgenic fish embryo, and any other type of transgenic fish cell or cluster of cells, whether haploid, diploid, triploid or other zygosity having at least one genomically integrated copy of a plasmid comprising a mutation target nucleic acid sequence. As used herein, the term xe2x80x9cembryoxe2x80x9d includes a single cell fertilized egg (i.e., a zygote) as well as a multicellular developmental stage of the organism. Preferably, the plasmid is integrated into the fish""s somatic and germ cells such that it is stable and inheritable. The transgenic fish or fish cell preferably contains a multiplicity of genomically integrated copies of the plasmid; more preferably, the multiple copies of the plasmid are integrated into the host organism""s genome in a contiguous, head-to-tail orientation. Progeny of the transgenic fish containing at least one genomically integrated copy of the plasmid, and transgenic fish derived from a transgenic fish egg, sperm, embryo or other fish cell of the invention, are also included in the invention. A fish is xe2x80x9cderived fromxe2x80x9d a transgenic fish egg, sperm cell, embryo or other cell if the transgenic fish egg, sperm cell, embryo or other cell contributes DNA to the fish""s genomic DNA. For example, a transgenic embryo of the invention can develop into a transgenic fish of the invention; a transgenic egg of the invention can be fertilized to create a transgenic embryo of the invention that develops into a transgenic fish of the invention; a transgenic sperm cell of the invention can be used to fertilize an egg to create a transgenic embryo of the invention that develops into a transgenic fish of the invention; and a transgenic cell of the invention can be used to clone a transgenic fish of the invention. In some preferred embodiments of the invention, the transgenic fish is sterile. The present invention further includes a cell line derived from a transgenic fish embryo or other transgenic fish cell of the invention, which contains at least one copy of a plasmid carrying an assayable mutation target nucleic acid sequence.
The mutation target nucleic acid sequence is preferably one having a characteristic or function, or encoding a gene product having a characteristic or function, that is detectably altered when mutated, thereby allowing the nonmutated form of the nucleic acid sequence to be distinguished from the mutated form. In a particularly preferred embodiment, a mutation in the mutation target nucleic acid sequence is detectable via bioassay in a bacterial cell, such as an E. coli cell, into which a mutation target nucleic acid sequence that has been isolated from the fish or fish cell has been introduced. In this regard, a transgenic fish having a triploid genome is desirable because triploidy allows larger amount of DNA to be recovered. An increase in the amount of DNA recovered has many advantages. For example, it allows for more efficient detection of the mutation target nucleic acid. Moreover, fish having a triploid genome are typically sterile, which may be desirable for certain applications or studies. The assayable mutation target nucleic acid sequence is typically heterologous with respect to the fish genome. Preferably, the plasmid is integrated into the host organism""s genome in a manner that avoids causing a detectable mutation in an endogenous gene of the host, thereby avoiding undesirably high background levels of mutation and reducing the sensitivity of the assay. The use of a smaller vector is preferred because the small size reduces the likelihood of physical disruption of one of the host cell""s genes. Preferred mutation target nucleic acid sequences include the lacI gene, the lacZ gene, the lac promoter sequence, and the rpsL gene. Preferably, the lacZ gene includes the lacZ promoter.
In another embodiment, the invention includes a genomically identical population of transgenic fish, each of whose somatic and germ cells contain at least one genomically integrated copy of a plasmid comprising an assayable mutation target nucleic acid sequence. The genomically identical population is a unisex population and can be male or female. Preferred embodiments of the genomically identical transgenic fish population are essentially as described for the transgenic fish of the invention. In an alternative embodiment, the invention includes a population of transgenic fish, i.e., an in-bred line, the members of which are not necessarily genomically identical but are homozygous with respect to genomically integrated plasmid.
Also provided is a method for mutation detection utilizing the transgenic fish or fish cell of the invention. This method is useful in evaluating the mutagenicity of various potential mutagens, such as chemical compounds, radioisotope emissions, and electromagnetic radiation. Mutations are detected in a mutation target nucleic acid sequence of a plasmid, wherein at least one copy of the plasmid has been integrated into the genomic DNA of the fish or fish cell. DNA containing the mutation target nucleic acid sequence is first recovered from the transgenic fish or fish cell, preferably by extracting the fish or fish cell DNA from the fish or fish cell, then cleaving the extracted DNA with a restriction endonuclease to yield at least one DNA fragment comprising the mutation target nucleic acid sequence derived from the plasmid, and multiple DNA fragments comprising chromosomal DNA. The DNA fragment that includes the mutation target nucleic acid sequence preferably includes substantially the entire plasmid, although it can contain a portion of the plasmid DNA, as long as it contains the mutation target nucleic acid sequence. Optionally, the method of mutation detection farther comprises separating the DNA fragment comprising the mutation target nucleic acid sequence from the multiple chromosomal fragments to isolate the mutation target DNA, although it should be understood. flat separation of the cleaved fragments comprising the mutation target DNA from the remaining chromosomal DNA is not required by the present method. If separation of the fragments is performed, the cleavage and the separation steps can be performed sequentially, or they can be performed simultaneously. In a particularly preferred embodiment of the method of the invention, wherein the mutation target nucleic acid sequence contains the lacZ gene, cleaved DNA fragments are separated by contacting the fragments with an affinity support comprising a lacZ operator binding material so as to immobilize the DNA fragment containing the mutation target nucleic acid sequence. After washing away the unbound DNA fragments, the bound DNA fragment is eluted from the support. The method further includes detection of the presence of a mutation in the mutation target nucleic acid sequence. Where the mutation target nucleic acid sequence contains the lacZ gene, mutations in the gene are preferably detected by transforming a host restriction-negative, lacZxe2x88x92xe2x88x92, galExe2x88x92 bacterial host with cleaved DNA comprising the mutation target nucleic acid sequence (whether or not the DNA fragments containing chromosomal DNA have been separated out); culturing the transformed bacteria on a lactose-containing or lactose analogue-containing medium; and selectively detecting a bacterial host that contains a mutation in the lacZ gene. Growth of the bacterial host is indicative of the existence of a mutation in the lacZ gene. Optionally, prior to transforming the bacterial host, the DNA comprising the mutation target nucleic acid test region is ligated to yield a circular DNA that is more efficiently electroporated. Also optionally, mutations in the mutation target nucleic acid sequence can be further analyzed, for example by nucleic acid sequence determination. When used to evaluate the mutagenicity of a particular agent, condition or event, the method further comprises, prior to extracting the fish DNA, exposing the transgenic fish or fish cell to the suspected mutagen.
The invention further includes a method for evaluating the mutagenicity of a suspected mutagen. A transgenic fish or fish cell of the invention is exposed to a suspected mutagen; the DNA containing the mutation target nucleic acid sequence is recovered from the transgenic fish or fish cell; and the presence of a mutation in the mutation target nucleic acid sequence is detected. Optionally, the mutated target nucleic acid sequence can be analyzed, for example by nucleic acid sequencing and the constructing a mutation spectrum.
Also included in the invention is a method for making a transgenic fish. Heterologous DNA is injected into a one-cell fish embryo, preferably through the micropyle, within about 10 minutes following fertilization, preferably within about 5 minutes following fertilization. A method for making a transgenic fish for mutagenesis detection includes microinjecting heterologous DNA into a one-cell fish embryo, wherein the heterologous DNA comprises a mutation target nucleic acid sequence, such as a lacZ gene.
The invention further includes a kit for detecting mutagenesis in transgenic fish comprising a genomically integrated plasmid comprising a mutation target nucleic acid. The kit includes, separately packaged, a host restriction-negative bacterial strain and a solid support that includes binding material capable of binding at least a portion of the plasmid. When used to detect mutagenesis in a transgenic fish having the lacZ gene as its mutation target nucleic acid sequence, the bacterial strain is preferably a host restriction-negative, lacZxe2x88x92, galExe2x88x92 E. coli strain, and the solid support preferably includes a lac operator binding material such as xcex2-galactosidase/LacI repressor fusion protein. The kit can optionally contain one or more additional components, such as a binding buffer to promote binding of the plasmid to the solid support, one or more restriction enzymes to excise the plasmid from the genomic DNA of the transgenic fish, an excision buffer, and a ligation for circularization of the excised plasmid prior to introduction of the plasmid into the bacterial host. The ligation buffer preferably contains a ligase, such as T4 ligase. The binding buffer and the excision buffer can, but need not be, the same buffer, so as to allow simultaneous excision of the plasmid and binding of the plasmid to the solid support.